Throughout this application various publications are referred to in square brackets by number. Full citations for these references may be found at the end of the specification. The disclosures of each of these publications, and also the disclosures of the patents and patent application publications recited herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
There is an urgent need for improved assays for both diagnosis of tuberculosis (TB) and drug susceptibility testing (DST) that are accurate, rapid and inexpensive [1]. The most important advances will be assays that can be applied at the point of care in the developing world. Recent advances in nucleic acid amplification approaches (“genotypic assays”), especially the Xpert MTB/RIF assay, are important contributions to the rapid initial diagnosis of pulmonary TB. However, Xpert MTB/RIF detects only rifampicin resistance (RifR) and relies on the fact that almost all clinical RifR identified to date is due to any of three specific point mutations [2-7]. Other clinical drug resistance phenotypes have more varied genetic underpinnings (e.g., hundreds of different mutations can lead to isoniazid resistance [8]) and are therefore refractory to diagnosis via sequence-specific approaches.
In South Africa both multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) strains are endemic; MDR-TB and XDR-TB together account for as much as 20% of all TB cases, and contribute significantly to mortality among hospitalized patients [9, 10]. Since both MDR-TB and XDR-TB are resistant to rifampicin, Xpert MTB/RIF cannot distinguish between them. Thus, because of the limitations of currently available diagnostic tests, patients with MDR-TB and XDR-TB are often put on inappropriate therapy for weeks or even months until drug susceptibility testing results become available.
Phenotypic assays, in contrast, recognize the organismal response of bacteria to antibiotics without limitation to any particular antibiotic, allele or mechanism. Culture remains the gold standard for phenotypic assays, but a classical culture identification of M. tuberculosis takes 4 to 8 weeks. Newer approaches, such as microscopic-observation drug-susceptibility (MODS), have shortened the time needed for phenotypic assays to between 1 and 2 weeks [11].
Fluorophages are a type of “reporter-phage” that inject their DNA specifically into mycobacteria [12]. Fluorescence is produced by expression of a fluorescent protein, such as Green Fluorescent Protein (GFP), gene cloned into the phage. Bacterial physiology required for growth and division is similar to that required to produce fluorescence, but the appearance of fluorescent signal after phage infection takes substantially less time than any assay requiring multiple cycles of cell growth and division. Moreover, the drugs which inhibit host gene expression likewise inhibit fluorescence. Since the proof of principle demonstration in 1993 [13], mycobacterial reporter phages have remained a potentially elegant solution to the problem of TB diagnosis. In laboratory cultures, including cultures derived from clinical isolates, reporter phages detect mycobacterial cells, and allow assays of DST in appreciably less time than culture alone [13-18]. However, existing reporter phages are unable to identify mycobacteria directly in clinical specimens, which limits their use.
The present invention address the need for improved mycobacterium phages for delivering nucleic acids of interest, including those encoding reporter proteins, into mycobacteria, and also addresses the need to identify mycobacteria directly in clinical specimens without costly equipment.